Cholecystokinin (CCK) is a classical gastrointestinal hormone produced from the proximal small intestine. It possesses a number of physiologic actions including regulation of pancreatic exocrine secretion, gallbladder contraction, gastric emptying, and perhaps satiety. Regulation of CCK secretion has been studied primarily by examining plasma levels of the hormone in animals and humans or in perfused intestinal segments. Although these methods are critical for understanding the physiology of CCK, they are limited in that they do not permit examination of the mechanisms that regulate CCK at the cellular level. The Principal Investigator has recently developed a method for isolating cells from the proximal small intestine in rats and enriching the CCK cells to 90% purity using a flow cytometer fluorescence-activated cell sorting technique. This method is based on the ability of monitor peptide (a CCK-releasing peptide) to selectively stimulate calcium fluxes in CCK cells and thereby provide a signal for separating CCK cells from a mixed population of intestinal cells loaded with the calcium-sensitive fluorescent dye, Indo-1 AM. These purified CCK cells retain viability and secrete CCK in response to specific secretagogues. The major advantage of this technique is that it provides a means of studying a single population of CCK cells, apart from other possible interfering influences such as neural, hormonal, or paracrine effects. The objectives of the proposed research plan are to use isolated CCK cells to (1) determine the neurotransmitters and hormones that directly regulate CCK secretion, and (2) characterize the second messenger pathways that mediate CCK release. These studies should provide insight into the cellular mechanisms that regulate CCK secretion.